Rongwei Guan

Gemcitabine eliminates double minute chromosomes from human ovarian cancer cells

Double minute chromosomes are cytogenetic manifestations of gene amplification frequently seen in cancer cells. Genes amplified on double minute chromosomes include oncogenes and multi-drug resistant genes. These genes encode proteins which contribute to cancer formation, cancer progression, and development of resistance to drugs used in cancer treatment. Elimination of double minute chromosomes, and therefore genes amplified on them, is an effective way to decrease the malignancy of cancer cells. We investigated the effectiveness of a cancer drug, gemcitabine, on the loss of double minute chromosomes from the ovarian cancer cell line UACC-1598. Gemcitabine is able to decrease the number of double minute chromosomes in cells at a 7500X lower concentration than the commonly used cancer drug hydroxyurea. Amplified genes present on the double minute chromosomes are decreased at the DNA level upon gemcitabine treatment. Gemcitabine, even at a low nanomolar concentration, is able to cause DNA damage. The selective incorporation of double minutes chromatin and γ-H2AX signals into micronuclei provides a strong link between DNA damage and the loss of double minute chromosomes from gemcitabine treated cells. Cells treated with gemcitabine also showed decreased cell growth, colony formation, and invasion. Together, our results suggest that gemcitabine is effective in decreasing double minute chromosomes and this affects the biology of ovarian cancer cells.

Differential expression of PAI-RBP1, C1orf142, and COTL1 in non-small cell lung cancer cell lines with different tumor metastatic potential.

Human non–small cell lung cancer (NSCLC) is one of the most common malignancies in the modern world. Its recurrence is mainly due to its ability to invade and metastasize. However, the precise mechanism for tumor development and metastasis is still not fully understood. To shed light on the development of lung cancer, the human giant cell lung carcinoma cell lines 95D with high metastatic potential and 95C with low metastatic potential were selected in this study. The 2 cell lines originated from the same parental cell and share a similar genetic background. In the current study, we identified 3 differentially expressed proteins in 95C and 95D cell lines, namely, PAI-RBP1, C1orf142, and COTL1, by using 2-dimensional electrophoresis proteomics analysis. We found that PAI-RBP1 and C1orf142 expression levels were higher in 95D than in 95C cells, whereas COTL1 expression level was lower in 95D when compared to 95C cells. We also confirmed these results by reverse transcription–polymerase chain reaction and immunoblotting analyses. The messenger RNA and protein levels of PAI-RBP1 and C1orf142 were much higher in 95D than in 95C cells, and COTL1 expression level was lower in 95D than in 95C cells. The PAI-RBP1 expression was assessed by immunohistochemistry in 70 NSCLC and 7 normal lung tissue samples from patients. PAI-RBP1 expression level was higher in tumor tissues (positive staining in 87.1% of cases [61/70]) than in normal tissues (positive staining in 14.3% of cases [1/7]). In conclusion, by studying protein expression in NSCLC cell lines with high and low metastasis as well as in human lung cancer tissues, we have identified 3 proteins, namely, PAI-RBP1, C1orf142, and COTL1, which were differentially expressed in NSCLC cell lines with different metastatic potential. In addition, we also found that PAI-RBP1 might contribute to NSCLC development.

Identification of Novel Subregions of LOH in Gastric Cancer and Analysis of the HIC1 and TOB1 Tumor Suppressor Genes in These Subregions

Previously, we identified 3 overlapping regions showing loss of heterozygosity (LOH, R1–R3 from 11 to 30 cM) on chromosome 17 in 45 primary gastric cancers (GCs). The data indicated the presence of tumor suppressor genes (TSGs) on chromosome 17 involved in GC. Among the putative TSGs in these regions, HIC1 (in SR1) and TOB1 (in SR3) remain to be examined in GC. By immunohistochemistry (IHC), methylation-specific PCR (MSP) and western blot, we evaluated the expression and regulation status for HIC1 and TOB1 protein in GC. We narrowed down the deletion intervals on chromosome 17 and defined five smaller LOH subregions, SR1–SR5 (0.54 to 3.42 cM), in GC. We found that HIC1 had downregulated expression in 86% (91/106) and was methylated in 87% (26/30) of primary GCs. Of the primary GCs showing downregulation of HIC1 protein, 75% (18/24) had methylated HIC1 gene. TOB1 was either absent or expressed at reduced levels in 75% (73/97) of the GC samples. In addition, a general reduction was found in total and the ratio of unphosphorylated to phosphorylated TOB1 protein levels in the differentiated GC cell lines. Further analysis revealed significant simultaneous downregulation of both HIC1 and TOB1 protein in GC tissue microarray samples (67%, 52/78) and in primary GCs (65%, 11/17). These results indicate that silencing of HIC1 and TOB1 expression is a common occurrence in GC and may contribute to the development and progression of the disease.

De novo-generated small palindromes are characteristic of amplicon boundary junction of double minutes

Double minutes (DMs) are hallmarks of gene amplification. However, their molecular structure and the mechanisms of formation are largely unknown. To elucidate the structure and underlying molecular mechanism of DMs, we obtained and cloned DMs using microdissection; and degenerated oligonucleotide primed polymerase chain reaction (DOP‐PCR) from the ovarian cancer cell line UACC‐1598. Two large amplicons, the 284 kb AmpMYCN, originating from locus 2p24.3 and the 391 kb AmpEIF5A2, from locus 3q26.2, were found co‐amplified on the same DMs. The two amplicons are joined through a complex 7 kb junction DNA sequence. Analysis of the junction has revealed three de novo created small palindromes surrounding the six breakpoints. Consistent with these observations, we further found that 70% of the 57 reported DM junction sequences have de novo creation of small palindromic sequences surrounding the breakpoints. Together, our findings indicate that de novo‐generated small palindromic sequences are characteristic of amplicon boundary junctions on DMs. It is possible that the de novo‐generated small palindromic sequences, which may be generated through non‐homologous end joining in concert with a novel DNA repair machinery, play a common role in amplicon rejoining and gene amplification.

Comparison of the inhibitory effects of three transcriptional variants of CDKN2A in human lung cancer cell line A549

BackgroundThe tumor suppressor gene CDKN2A generates at least three different transcriptional variants, each of which is thought to encode a tumor suppressor. However, the inhibitory activities of these variants have not yet been compared in the same cells. Protein therapy is known to have several advantages over gene therapy. Thus, investigation of the exogenous protein molecule of the most effective suppressor may yield meaningful information regarding protein-based cancer therapy.MethodsThe inhibitory effects of p16INK4a, p14ARF and p12 were studied in the human lung cancer cell line A549 which lacks the CDKN2A locus. The eukaryotic expression plasmids of the three transcriptional variants were constructed and stably transfected into the cells. RNA and protein expression by the plasmids was confirmed using RT-PCR and fluorescence immunocytochemistry, respectively. Cell growth inhibition and cell-cycle redistribution after transfection were investigated based on growth curve and flow cytometry analyses. An exogenous His-tag fusion p16INK4a protein was obtained and purified by affinity chromatography. Cell growth inhibition and cell cycle arrest induced by the expression of p16INK4a protein were measured in A549 cells transduced with the exogenous protein.ResultsWhile all three variants suppressed cell growth, p16INK4a had the strongest effect. Marked G1-phase accumulation and S-phase inhibition were induced by p16INK4a and p14ARF but not by p12. Exogenous p16INK4a protein was successfully expressed and purified and transduction of the fusion protein into A549 cells inhibited cell growth by G1→S arrest.ConclusionsAmong the three transcript variants, p16INK4a has a greater inhibitory effect than p14ARF and p12; exogenous p16INK4a protein should be further investigated for use in cancer therapy as a protein agent.

Expulsion of micronuclei containing amplified genes contributes to a decrease in double minute chromosomes from malignant tumor cells

Double minute chromosomes (DMs) are a hallmark of gene amplification. The relationship between the formation of DMs and the amplification of DM‐carried genes remains to be clarified. The human colorectal cancer cell line NCI‐H716 and human malignant primitive neuroectodermal tumor cell line SK‐PN‐DW are known to contain many DMs. To examine the amplification of DM‐carried genes in tumor cells, we performed Affymetrix SNP Array 6.0 analyses and verified the regions of amplification in NCI‐H716 and SK‐PN‐DW tumor cells. We identified the amplification regions and the DM‐carried genes that were amplified and overexpressed in tumor cells. Using RNA interference, we downregulated seven DM‐carried genes, (NDUFB9, MTSS1, NSMCE2, TRIB1, FAM84B, MYC and FGFR2) individually and then investigated the formation of DMs, the amplification of the DM‐carried genes, DNA damage and the physiological function of these genes. We found that suppressing the expression of DM‐carried genes led to a decrease in the number of DMs and reduced the amplification of the DM‐carried genes through the micronuclei expulsion of DMs from the tumor cells. We further detected an increase in the number of γH2AX foci in the knockdown cells, which provides a strong link between DNA damage and the loss of DMs. In addition, the loss of DMs and the reduced amplification and expression of the DM‐carried genes resulted in a decrease in cell proliferation and invasion ability.

RPL13A as a reference gene for normalizing mRNA transcription of ovarian cancer cells with paclitaxel and 10-hydroxycamptothecin treatments.

Gene transcription analysis is important in cancer research, and reverse transcription‑quantitative polymerase chain reaction (RT‑qPCR) has been demonstrated to be an effective method to evaluate gene transcription in cancer. RT‑qPCR requires an internal reference gene with a consistent level of mRNA transcription across various experimental conditions. However, it has been suggested that different treatments, including anticancer therapy, may influence the transcriptional stability of internal reference genes. Paclitaxel (PTX) and 10‑hydroxycamptothecin (HCPT) are widely used to treat various types of cancer, and a suitable internal reference gene is required in order to analyze the transcription profiles of the cells following treatment. In the current study, the transcriptional stability of 30 candidate reference genes was investigated in cancer cells following treatment with PTX and HCPT. The two ovarian cancer cell lines, UACC‑1598 and SKOV3, were treated with PTX and HCPT for 24 and 48 h, and the transcriptional levels of the candidate reference genes were subsequently evaluated by RT‑qPCR analysis. The transcriptional stability of the selected genes was then analyzed using qbase+ and NormFinder software. A total of 9 genes were demonstrated to exhibit high transcriptional stability and one of these genes, ribosomal protein L13a (RPL13A), was identified to exhibit high transcriptional stability in every group. The current study identified various reference genes suitable under different circumstances, while RPL13A was indicated to be the most suitable reference gene for analyzing the transcription profile of ovarian cancer cells following treatment with PTX and HCPT.

Combinational analysis of linkage and exome sequencing identifies the causative mutation in a Chinese family with congenital cataract

BackgroundCongenital cataract is a Mendelian disorder that frequently causes blindness in infants. To date, various cataract-associated loci have been mapped; more than 30 genes have been identified by linkage analysis. However, the pathogenic loci in some affected families are still unknown, and new research strategies are needed. In this study, we used linkage-exome combinational analysis to further investigate the pedigree of a four-generation Chinese family with autosomal dominant coralliform cataract.MethodsWe combined whole exome sequencing and linkage analysis to identify the causative mutation. The exome capture and next-generation sequencing were used to sequence the protein-coding regions in the genome of the proband to identify rare mutations, which were further screened for candidate mutations in linkage regions. Candidate mutations were independently verified for co-segregation in the whole pedigree using Sanger sequencing.ResultsWe identified a C to A transversion at nucleotide position c.70 in exon 2 of CRYGD, a cataract-associated gene. This mutation resulted in a threonine substitution for proline at amino acid residue 24.ConclusionsWe identified a missense P24T mutation in CRYGD that was responsible for coralliform cataract in our studied family. Our findings suggest that the combination of exome sequencing and linkage analysis is a powerful tool for identifying Mendelian disease mutations that might be missed by the classic linkage analysis strategy.

Overexpression of RCC2 Enhances Cell Motility and Promotes Tumor Metastasis in Lung Adenocarcinoma by Inducing Epithelial–Mesenchymal Transition

Purpose: Investigate the role of regulator of chromosome condensation 2 (RCC2) on lung adenocarcinoma (LUAD) metastasis. Experimental Design: Clinical specimens were used to assess the impact of RCC2 on LUAD metastasis. Mouse models, cytobiology, and molecular biology assays were performed to elucidate the function and underlying mechanisms of RCC2 in LUAD. Results: RCC2 expression was frequently increased in LUADs (88/122, 72.13%). It was confirmed by analysis of a larger cohort of TCGA RNA-seq data containing 488 LUADs and 58 normal lung tissues (P < 0.001). Importantly, increased level of RCC2 was significantly associated with T status of tumor (P = 0.002), lymph node metastasis (P = 0.004), and advanced clinical stage (P = 0.001). Patients with LUAD with higher expression of RCC2 had shorter overall survival. Cox regression analysis demonstrated that RCC2 was an independent poorer prognostic factor for patients with LUAD. Moreover, forced expression of RCC2 promoted intrapulmonary metastasis in vivo and significantly enhanced LUAD cell migration, invasion, and proliferation in vitro. Further study found that RCC2 induced epithelial–mesenchymal transition (EMT) and also stimulated the expression of MMP-2 and MMP-9. In addition, RCC2 was able to activate JNK, while inhibition of JNK suppressed the effect of RCC2 on LUAD cell migration, invasion, EMT, and the expression of MMP-2 and MMP-9. Conclusions: RCC2 plays a pivotal role in LUAD metastasis by inducing EMT via activation of MAPK–JNK signaling. Clin Cancer Res; 23(18); 5598–610. ©2017 AACR.

Lack of association between thyroid-stimulating hormone receptor haplotypes and Graves' disease in a northern Chinese population.

Graves’ disease is a common autoimmune thyroid disease affecting 0.5%–1.0% of the general population. Genetic and environmental factors play a role in the pathogenesis of Graves’ disease. Thyroid-stimulating hormone receptor (TSHR) represents the primary auto-antigen in Graves’ disease, causing the characteristic clinical phenotype (1, 2). The TSHR gene is an important immunoregulatory factor and has long been thought to be a candidate gene for Graves’ disease. Some single-nucleotide polymorphisms (SNPs) of the TSHR gene have been identified a key role in triggering the onset of Graves’ disease. Recently, many researchers focus on the association between the SNPs in intron 1 of the TSHR gene and Graves’ disease. In 2005, Dechairo et al. found that the strongest association SNP was rs2268458 in a large Caucasian cohort (3). Brand et al. found that the SNPs with the strongest association were rs179247 and rs12101255 in European populations (4). For a case-control cohort Chinese study, we selected 199 Graves’ disease patients who were patients at the Department of Endocrinology in the Second Affiliated Hospital of Harbin Medical University, China. A total of 208 control subjects were recruited from the Heilongjiang Hospital and Central Blood Bank of Heilongjiang Province, China. All control subjects were age and sex matched with the patients. The study was approved by the Institutional Ethics Committee. In summary, our results provide preliminary evidence that the three SNPs (rs179247, rs12101255 and rs2268458) are not involved in the pathogenesis of Graves’ disease in a northern Chinese population. None of the three SNPs was observed to be significantly associated with Graves’ disease, and none of the eight haplotypes showed significant association with Graves’ disease in this population (P > 0.05). The results of the present study should be interpreted with caution for the limited numbers of samples. Genetic studies combined with further functional studies will be required to validate any association and to understand any mechanism conferred by SNPs of the TSHR gene to Graves’ disease.